Purpose:
Diabetic retinopathy is a common blinding disease characterised by the progressive degeneration of retinal vessels. Like in other vascular diseases, the insufficiency of the currently applied treatments imposes the need for new therapeutic approaches. The discovery of endothelial progenitor cells (EPCs) -shown to contribute to vascular repair- revealed the potential for cell-based therapies that will target the pathophysiological mechanisms of vascular diseases. However, little is known about the origin, phenotypic and functional characteristics of EPCs. With this study we aimed to establish a protocol for the isolation and phenotypic characterisation of circulating EPCs (cEPCs) and to identify “key” genes expressed by the EPC-enriched fraction of CD34+CD45low cells.

Methods:
Using an optimized method for flow cytometry analysis, cEPCs -defined as CD34+CD45low - were isolated from the fraction of peripheral blood mononuclear cells. For the phenotypic characterisation of these cells qPCR analysis was performed using as control population the CD34-CD45high myelo/monocytic cells. The list of genes that were tested included the surface molecules CD34, CD45, CD133, CD309, CD14, CXCR4, Brachyury (Br), CD117/c-kit and VE-Cadherin and the transcription factors GATA2 and Scl/Tal-1.

Results:
The comparative analysis of gene expression showed that the cEPC-enriched fraction strongly expressed CD133 but not CD309. The mesenchymal transcription factor GATA2 had also a significantly higher expression in the population of putative cEPCs. Three independent series of experiment were performed and all agreed with the above results. No difference in the expression of the other genes tested was identified between the two populations.

Conclusions:
Our results suggest that the CD34+CD133+ phenotype is more efficient for the isolation of cEPCs compare to the CD34+CD309+ combination. Also, the expression of GATA2 supported the existence of circulating adult hemangioblasts which like their embryonic counterparts retain the potential for differentiation into hematopoietic as well as endothelial cells. Finally, this study demonstrated that the combination of flow cytometric and qPCR analysis is a useful approach for the isolation and genotypic profiling of cEPCs.